This invention relates generally to a freezing apparatus and more particularly to a high pressure freezing apparatus for preserving biological samples.
It is well-known in the medical arts that to examine biological samples and determine the cellular structure and function thereof, the samples must be “fixed” with minimal alteration of the structural integrity of the cells making up the sample. Typically, freezing machines are used to freeze a biological sample to preserver the structural integrity of the sample. The biological samples to be examined may include samples suspended in an solution as well as human or animal tissue segments and entire organs for transplant or virtually any other material that is desirable for study and examination of cellular structure. The freezing machines create a zone of freezing as thick as possible in the sample so that the cellular structure is preserved in a state that promotes the examination thereof.
When ice crystals form in a living cell or tissue they first extract the water from the cell and form pure ice with nothing dissolved in them (e.g., they form as “snowflakes”). The ice crystal formation not only physically “stabs” the cell to death, but it compresses all its living molecules into narrower and narrower interstices between the growing snowflakes. The compressed molecules are subjected to very abnormal salt and electrolyte compositions and very abnormal packing, resulting in gross distortion of cellular structure, and ultimately, by the end of the freezing process, in cell death. To avoid the gross mechanical deformations that occur to cells and to the molecules within them when their water is converted to crystalline ice, the zone of freezing in a sample to be examined should be accomplished at high pressures (at least 2000 bar) so that water in the sample cannot expand or crystallize as it freezes.
One existing freezing machine, sold under the trade name CRYOPRESS by MedVac Inc. of St. Louis, Mo., uses gravity to “slam” a biological sample onto an ultra-cold block of metal cooled to minus 177 degrees C., the temperature of liquid nitrogen, or to minus 269 degrees C., liquid helium temperature. Another earlier machine designed by others and sold by Lifecell, Inc., of San Antonio, Tex., uses a pneumatic cylinder to slam the sample onto the ultra-cold block. The basic limitation of all such devices is that they properly freeze only a very thin layer (about 5-10 microns) at the surface of the sample that strikes the ultra cold metal block. Also, all such existing “freeze-slammers” incorporate spring-dampers to prevent the sample from bouncing off the cold metal block. Frequently, the dampers do not effectively prevent bounce resulting in non-uniform freezing in the sample.
More importantly, none of these devices are designed to apply pressure to the sample as it strikes the cryo-block. On the contrary, they are designed to absorb pressure so as to prevent bounce (as mentioned above). Current designs of high-pressure freezing machines rely on injecting a cryogenic fluid into a vessel containing the sample, in order to accomplish freezing during the application of high pressure. However, these designs freeze relatively slowly, because a liquid cryogen is applied to the sample rather than a metallic surface that is held at cryogenic temperatures, which is much more conductive of heat. Other shortcomings of existing high-pressure freezing machines include the fact that they are expensive to manufacture and are difficult to operate, and are highly unreliable as a result of the complex mechanical processes that are required to generate and maintain elevated pressures with cryogenic liquids.
Therefore, a need exists for a high-pressure freezing machine that is dependable, inexpensive to manufacture and operate, and that both freezes as fast as possible and sustains as much pressure as possible, in order to produce the best possible freezing.